New copolymers of N,N-dimethylacrylamide with blocked isocyanates for oligonucleotide immobilization in DNA microarray technology

A family of copolymers of N,N-dimethylacrylamide containing blocked isocyanate functionalities is presented. The copolymers were characterized by DSC, GPC and ¹H NMR. Calorimetric analysis showed for any composition broad endothermal phenomena followed by a stronger exothermal peak, which can be attributed, respectively, to the deblocking and subsequent reaction of generated NCO groups.Characterization of glass slides coated with these polymers was done by contact angle measurements and atomic force microscopy. While the former method revealed minimal differences with formation of moderately hydrophilic surfaces, microscopic images showed a more homogeneous coating formation for the copolymer structure with 50% molar of blocked isocyanate. The efficiency of the coated substrates in the immobilization of amino functionalized oligonucleotides was successfully assessed through binding tests and analysis by confocal fluorescence microscopy.Finally, some model microarrays were fabricated by spotting and hybridization with complementary, fluorescently labelled targets was carried out. It resulted in surfaces coated with copolymers which show well defined circular spots with a fluorescence intensity higher than that obtained by slides treated by silanization, and based on the same immobilization chemistry.     

DNA nanotechnology for nucleic acid analysis: DX motif-based sensor

A light on the tiles: A sensor that fluoresces in the presence of specific nucleic acids was designed and characterized. The sensor uses a molecular beacon probe and three adaptor strands to form a five-stranded assembly, a DX-tile, with a specific analyte. This sensor is a highly selective and affordable tool for the real-time analysis of DNA and RNA.     

An Optimized Protocol for DNA Extraction from Wheat Seeds and Loop-Mediated Isothermal Amplification (LAMP) to Detect Fusarium graminearum Contamination of Wheat Grain

A simple, rapid, and efficient method for isolating genomic DNA from germinated seeds of wheat that is free from polysaccharides and polyphenols is reported. DNA was extracted, treated with RNase, measured and tested for completeness using agarose gel electrophoresis. DNA purification from wheat grains yielded abundant, amplifiable DNA with yields typically between 100 and 200 ng DNA/mg. The effectiveness and reliability of the method was tested by assessing quantity and quality of the isolated DNA using three PCR-based markers. Inter-simple sequence repeats (ISSRs) were used to assess the genetic diversity between different wheat varieties. Specific PCR primer pair Tox5-1/Tox5-2 and a loop-mediated isothermal amplification (LAMP) procedure were used to detect genomic DNA of Fusarium graminearum in contaminated wheat seeds. In this method there is no need to use liquid nitrogen for crushing germinated seedlings. The protocol takes approximately one hour to prepare high quality DNA. In combination with the LAMP assay it is a fast and cost-effective alternative to traditional diagnostic methods for the early detection of toxigenic fusaria in cereals.     

A DNA electrochemical sensor with poly-l-lysine/single-walled carbon nanotubes films and its application for the highly sensitive EIS detection of PAT gene fragment and PCR amplification of NOS gene

A sensitive and novel DNA electrochemical biosensor for the detection of the transgenic plants gene fragment by electrochemical impedance spectroscopy (EIS) was presented. The well-dispersed carboxylic group-functionalized single-walled carbon nanotubes (SWNTs) were dripped onto the carbon paste electrode (CPE) surface firstly, and poly-l-lysine films (pLys) were subsequently electropolymerized by cyclic voltammetry (CV) to prepare pLys/SWNTs/CPE. The morphology of pLys/SWNTs films was examined using a field emission scanning electron microscope (SEM). The pLys/SWNTs films modified electrode exhibited very good conductivity. DNA probes were easily immobilized on the poly-l-lysine films via electrostatic adsorption. The hybridization events were monitored with electrochemical impedance spectroscopy using [Fe(CN)₆]^3−/4− as indicator. The PAT gene fragment from phosphinothricin acetyltransferase gene was detected by this DNA electrochemical sensor. The dynamic detection range of this sensor to the PAT gene fragment was from 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ mol/L. A detection limit of 3.1 × 10⁻¹³ mol/L could be estimated. The PCR amplification of NOS gene from the sample of a kind of transgenic modified bean was also detected satisfactorily by EIS.     

Specimen preparation and image processing and analysis techniques for automated quantification of concrete microcracks and voids

Specimen preparation and image processing/analysis techniques were developed for use in automated quantitative microstructural investigation of concrete, focusing on concrete microcracks and voids. Different specimen preparation techniques were developed for use in fluorescent and scanning electron microscopy (SEM) of concrete; then techniques produce a sharp contrast between microcracks/voids and the body of concrete. The image processing/analysis techniques developed specifically for use with concrete address the following usages: automatic threshold; development of intersecting microcracks/voids and connected voids; distinction of microcracks form voids based on geometric attributes; and noise filtration.     

A highly efficient method for concentrating DNA from river water by combined coagulation and foam separation

ABSTRACT

Both Escherichia coli and Enterococci were collected in foam within 7 min from 500 mL of bacteria-spiked water by coagulation and foam separation using ferric chloride and milk casein. These bacterial DNA isolated in the 100 µL of extract from the foam more than 87.5% recovery using the DNeasy PowerWater® Kit. To test this method with water from three natural rivers, 0.67–2.70 µg of DNA were concentrated in 100 µL of extract from 1,000 mL of river water. When the DNA extract was subjected to 16S rRNA gene sequencing analysis, information on the bacterial flora could be obtained.